Question

Many bacterial genes involved in amino acid biosynthesis are regulated by RNA leaders that respond, indirectly, to the level of a specific amino acid. Like attenuators or some riboswitches, these allosteric RNA leaders, called $T$ -box leaders, can form either terminators or antiterminators. The name $T$ -box refers to a 14 -nucleotide sequence present in all of these RNA leaders; a $5^{\prime}$ UGGU $3^{\prime}$ sequence within the T-box is complementary to the conserved $3^{\prime}$ end of tRNAs $\left(5^{\prime} \text { ACCA } 3^{\prime}\right)$ The first T-box RNA device was discovered in the $B$. subtilis tyrS gene, which encodes tyrosyl-tRNA synthetase, the enzyme that charges tRNA $^{\text {Tyr }}$ with tyrosine. As shown in the following diagram, the T-box leader can bind to the anticodon, and at the same time, to the $3^{\prime}$ end of the same uncharged tRNA $^{\text {Tyr }}(\text { left })$. When uncharged tRNA $^{\text {Tyr is bound, an }}$ antiterminator forms in the leader; otherwise, a terminator forms (right). a. Most of the tRNA synthetase genes in $B$. subtilis are regulated by T-box RNA leaders that respond to specific uncharged tRNAs. Explain the logic of this regulation. b. How could you alter the base sequence of the B. subtilis tyrS T-box leader so that it might respond to uncharged tRNA $^{\text {Phe instead of uncharged t} R N A^{\text {Tyr }} \text { ? }}$ c. The T-box is responsible for nearly all of the regulation of tyrS gene expression. What do you predict would happen to tyrS gene expression if the the $5^{\prime}$ UAC $3^{\prime}$ in the RNA leader that interacts with the tRNA $^{\text {Tyr anticodon was changed to to } 5^{\prime} \text { CUA } 3^{\prime} ?}$ Explain. L. Key experimental support for the idea that the T-box RNA leader binds tRNAs was the finding that normal function could be restored to the mutant T-box described in part (c) by a particular mutation in a gene different from tyrS. What specific bacterial gene mutation would render the mutant T-box RNA leader functional again? Results of experiments involving a tyrS-lacZ reporter gene indicated that the $t y r S$ T-box leader responds directly to relative levels of charged versus uncharged tRNAs rather than to the availability of tyrosine. These experiments involved expression of mutant tRNA $^{\text {Tyr }}$ species. These mutations were outside of the anticodon or the four base pairs at the $3^{\prime}$ end, yet they prevented the tRNA from being charged by tRNA synthetase. e. An otherwise wild-type $B$. subtilis strain contains a tyrS -lacZ reporter transgene that includes the T-box RNA leader. Compare the expression of $\beta$ -galactosidase in this strain when tyrosine levels are low as opposed to when tyrosine levels are high. f. Suppose now that the $t R N A^{T y r}$ gene in the strain described in part (e) was replaced with the mutant $t R N A^{T y r}$ gene described earlier with mutations that prevented the tRNA from being charged. Compare the expression of $\beta$ -galactosidase in this new strain when tyrosine levels are low as opposed to when tyrosine levels are high. Explain how this experiment distinguished the two hypotheses. g. T-box regulators have been identified in many other bacterial species using computer algorithms. What do you think the computer programs searched for?

Name: Problem 40, Chapter 16: Genetics: From Genes to Genomes
Uploaded: 2021-10-27T06:25:36-08:00
Duration: 4 min 59 s
Channel: Sana Riaz

   Many bacterial genes involved in amino acid biosynthesis are regulated by RNA leaders that respond, indirectly, to the level of a specific amino acid. Like attenuators or some riboswitches, these allosteric RNA leaders, called $T$ -box leaders, can form either terminators or antiterminators. The name $T$ -box refers to a 14 -nucleotide sequence present in all of these RNA leaders; a $5^{\prime}$ UGGU $3^{\prime}$ sequence within the T-box is complementary to the conserved $3^{\prime}$ end of tRNAs $\left(5^{\prime} \text { ACCA } 3^{\prime}\right)$

The first T-box RNA device was discovered in the $B$. subtilis tyrS gene, which encodes tyrosyl-tRNA synthetase, the enzyme that charges tRNA $^{\text {Tyr }}$ with tyrosine. As shown in the following diagram, the T-box leader can bind to the anticodon, and at the same time, to the $3^{\prime}$ end of the same uncharged tRNA $^{\text {Tyr }}(\text { left })$. When uncharged tRNA $^{\text {Tyr is bound, an }}$ antiterminator forms in the leader; otherwise, a terminator forms (right).
a. Most of the tRNA synthetase genes in $B$. subtilis are regulated by T-box RNA leaders that respond to specific uncharged tRNAs. Explain the logic of this regulation.
b. How could you alter the base sequence of the
B. subtilis tyrS T-box leader so that it might respond to uncharged tRNA $^{\text {Phe instead of uncharged t} R N A^{\text {Tyr }} \text { ? }}$
c. The T-box is responsible for nearly all of the regulation of tyrS gene expression. What do you predict would happen to tyrS gene expression if the the $5^{\prime}$ UAC $3^{\prime}$ in the RNA leader that interacts with the tRNA $^{\text {Tyr anticodon was changed to to } 5^{\prime} \text { CUA } 3^{\prime} ?}$ Explain.
L. Key experimental support for the idea that the T-box RNA leader binds tRNAs was the finding that normal function could be restored to the mutant T-box described in part (c) by a particular mutation in a gene different from tyrS. What specific bacterial gene mutation would render the mutant T-box RNA leader functional again?
Results of experiments involving a tyrS-lacZ reporter gene indicated that the $t y r S$ T-box leader responds directly to relative levels of charged versus uncharged tRNAs rather than to the availability of tyrosine. These experiments involved expression of mutant tRNA $^{\text {Tyr }}$ species. These mutations were outside of the anticodon or the four base pairs at the $3^{\prime}$ end, yet they prevented the tRNA from being charged by tRNA synthetase.
e. An otherwise wild-type $B$. subtilis strain contains a tyrS -lacZ reporter transgene that includes the
T-box RNA leader. Compare the expression of $\beta$ -galactosidase in this strain when tyrosine levels are low as opposed to when tyrosine levels are high.
f. Suppose now that the $t R N A^{T y r}$ gene in the strain described in part (e) was replaced with the mutant $t R N A^{T y r}$ gene described earlier with mutations that prevented the tRNA from being charged. Compare the expression of $\beta$ -galactosidase in this new strain when tyrosine levels are low as opposed to when tyrosine levels are high. Explain how this experiment distinguished the two hypotheses.
g. T-box regulators have been identified in many other bacterial species using computer algorithms. What do you think the computer programs searched for?

Genetics: From Genes to Genomes

Leland Hartwell,… 6th Edition

Chapter 16, Problem 40

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Solved by Expert Sana Riaz

10/27/2021

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subtilis. The T-box RNA leaders regulate gene expression based on the availability of uncharged tRNAs. When the specific uncharged tRNA (e.g., tRNA^Tyr) accumulates, it binds to the T-box leader, forming an antiterminator structure that allows transcription of the Show more…

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Many bacterial genes involved in amino acid biosynthesis are regulated by RNA leaders that respond, indirectly, to the level of a specific amino acid. Like attenuators or some riboswitches, these allosteric RNA leaders, called $T$ -box leaders, can form either terminators or antiterminators. The name $T$ -box refers to a 14 -nucleotide sequence present in all of these RNA leaders; a $5^{\prime}$ UGGU $3^{\prime}$ sequence within the T-box is complementary to the conserved $3^{\prime}$ end of tRNAs $\left(5^{\prime} \text { ACCA } 3^{\prime}\right)$ The first T-box RNA device was discovered in the $B$. subtilis tyrS gene, which encodes tyrosyl-tRNA synthetase, the enzyme that charges tRNA $^{\text {Tyr }}$ with tyrosine. As shown in the following diagram, the T-box leader can bind to the anticodon, and at the same time, to the $3^{\prime}$ end of the same uncharged tRNA $^{\text {Tyr }}(\text { left })$. When uncharged tRNA $^{\text {Tyr is bound, an }}$ antiterminator forms in the leader; otherwise, a terminator forms (right). a. Most of the tRNA synthetase genes in $B$. subtilis are regulated by T-box RNA leaders that respond to specific uncharged tRNAs. Explain the logic of this regulation. b. How could you alter the base sequence of the B. subtilis tyrS T-box leader so that it might respond to uncharged tRNA $^{\text {Phe instead of uncharged t} R N A^{\text {Tyr }} \text { ? }}$ c. The T-box is responsible for nearly all of the regulation of tyrS gene expression. What do you predict would happen to tyrS gene expression if the the $5^{\prime}$ UAC $3^{\prime}$ in the RNA leader that interacts with the tRNA $^{\text {Tyr anticodon was changed to to } 5^{\prime} \text { CUA } 3^{\prime} ?}$ Explain. L. Key experimental support for the idea that the T-box RNA leader binds tRNAs was the finding that normal function could be restored to the mutant T-box described in part (c) by a particular mutation in a gene different from tyrS. What specific bacterial gene mutation would render the mutant T-box RNA leader functional again? Results of experiments involving a tyrS-lacZ reporter gene indicated that the $t y r S$ T-box leader responds directly to relative levels of charged versus uncharged tRNAs rather than to the availability of tyrosine. These experiments involved expression of mutant tRNA $^{\text {Tyr }}$ species. These mutations were outside of the anticodon or the four base pairs at the $3^{\prime}$ end, yet they prevented the tRNA from being charged by tRNA synthetase. e. An otherwise wild-type $B$. subtilis strain contains a tyrS -lacZ reporter transgene that includes the T-box RNA leader. Compare the expression of $\beta$ -galactosidase in this strain when tyrosine levels are low as opposed to when tyrosine levels are high. f. Suppose now that the $t R N A^{T y r}$ gene in the strain described in part (e) was replaced with the mutant $t R N A^{T y r}$ gene described earlier with mutations that prevented the tRNA from being charged. Compare the expression of $\beta$ -galactosidase in this new strain when tyrosine levels are low as opposed to when tyrosine levels are high. Explain how this experiment distinguished the two hypotheses. g. T-box regulators have been identified in many other bacterial species using computer algorithms. What do you think the computer programs searched for?

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Key Concepts

Computational Identification of Regulatory RNA Motifs

Computational identification of regulatory RNA motifs involves using algorithms to scan genomic sequences for conserved RNA structures and sequence elements indicative of regulatory functions. These bioinformatics tools are critical for discovering new classes of regulatory RNAs across diverse organisms by identifying sequences that form characteristic secondary structures associated with gene regulation. This approach helps expand our understanding of the complexity and diversity of RNA-mediated control mechanisms.

Regulatory RNA Elements

Regulatory RNA elements are segments of RNA that influence gene expression without being translated into proteins. They do this by adopting specific secondary structures that can bind small molecules, metabolites, or even other RNAs, thereby changing their structure and function. This mechanism is seen in riboswitches and other RNA-based regulatory systems where binding events control downstream gene expression, typically at the level of transcription termination or translation initiation.

Mechanism of Transcription Attenuation

Transcription attenuation is a regulatory process where the formation of alternative RNA secondary structures in the leader region of a transcript determines whether transcription will continue or terminate prematurely. Specific structured regions, such as terminators or antiterminators, form depending on cellular conditions, including the presence or absence of certain ligands or binding partners, thus modulating gene expression in response to metabolic needs.

tRNA Charging and Sensing

tRNA charging refers to the attachment of an amino acid to its corresponding tRNA by a tRNA synthetase, which is essential for protein synthesis. In many regulatory systems, the ratio of charged to uncharged tRNA serves as an indicator of amino acid availability within the cell. Regulatory RNAs can sense the charging state and thereby modulate gene expression by adopting alternative structures based on whether they interact with charged or uncharged tRNA molecules.

RNA-RNA Base Pairing Specificity

RNA-RNA base pairing specificity is a fundamental principle that underlies the interaction between different RNA molecules. In regulatory mechanisms, complementary sequences allow regulatory RNAs to precisely recognize and bind to specific tRNAs or other RNAs. This specificity of interaction ensures that the regulatory outcomes are fine-tuned to respond to the correct signals, such as particular amino acid deficits or metabolic conditions.

Reporter Gene Assays in Gene Regulation Studies

Reporter gene assays involve fusing a regulatory region of interest to a reporter gene, such as lacZ, enabling researchers to monitor regulatory activity by measuring reporter gene expression. These assays provide a quantitative and visible means to assess how changes in the regulatory elements or cellular conditions affect gene expression. This is particularly useful in dissecting the mechanisms of RNA-based regulation and understanding the impact of specific mutations.

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